Biological and clinical significance of tTG and isopeptide in AD Abstract: Extensive protein crosslinkings and aggregation is one of the most important molecular events associated with the pathogenesis of Alzheimer's disease (AD). During AD development, intra- and extracellular proteins are cross-linked, leading to neuronal loss and progressive decline in cognition. Identifing substrate(s) for declined cognition is important not only for eventually solving the mystery of pathogenic mechanism of AD, but also for designing effective therapeutic reagents for the prevention and treatment of AD. Because tissue transglutaminase (tTG) catalyzes the cross-linking of a spectrum of critical proteins including tau, beta-amyloid, neurofilament and alpha-synuclein that are major components of neurofibrillary tangles or neuritic plaques in AD brains, we hypothesize that increased tTG and/or tTG enzymatic activity may initiate protein cross- linking, and the accumulation of cross-linked protein measured by the level of iso-peptide may be closely correlated with the declining mental status of AD patients during the pathogenesis. To test this hypothesis, we propose three Specific Aims: 1. To study the temporal and regional levels of tTG as a function of the development of AD. The mRNA, protein and enzymatic activity of tTG will be measured in cortical gray matter from multiple brain regions including frontal, temporal, pariteal and occipital lobes of postmortem brains from AD, mild cognitive impairment (MCI) and age-matched normal controls (NCI). A group of young normal brains and a group of Parkinson's disease (PD) brains will also be included in this study as controls. AD, MCI, NCI and PD brain samples will be provided from the Brain Bank of prospective longitudinal Religious Order Study using quantitative immunochemical methods. Brain samples of non-aged normal controls (NANC) will be provided by UWHC/VAH and Mayo Clinic Jacksonville Brain Banks. 2. To study changes of isopeptide that is the major product of tTG activity during the AD pathogenesis. Levels of isopeptide from the same panel of samples used in Aim 1 will be quantified with immunochemical methods. 3. To characterize the status of isopeptide and its relationship to clinical neuropsychological performance with the emphasis on the "normal" aging cases and MCI cases. tTG protein, enzymatic activity, iso-peptide level and relevant clinical cognitive or neuropathological parameters will be correlated respectively to explore if accumulation of iso-peptide, presumably due to transglutaminase activity in human brains is an important contributing factor to progressive cognitive declining. If the data obtained from this R21 proposal support our major hypothesis, we will formulate a R01 application to characterize the major substrate(s) that harbor isopeptide. Because the major substrates with isopeptide may vary depending upon the pathogenic stages of AD, some major substrates in the early stage of the disease may be gradually replaced by others in the later stage. Identification of these substrates, especially in the early stages will provide important information regarding AD pathogenesis and help to design preventive and therapeutic reagents for this devastating disease. PUBLIC HEALTH RELEVANCE: This grant application intends to investigate the clinical and biological effects of tissue transglutaminase (tTG) and the accumulation of its product, isopeptides, on the decline of mental status during the pathogenesis of AD in a group of patients collected from Religious Order Study. This will be first systemic study to investigate the relationship of tTg and its product-isopeptides and the cognitive status during the development of AD. The positive data obtained from this R21 project will lead to a new R01 project that will characterize the major substrate(s) that harbor isopeptides in the postmortem human brains. Identification of these substrates, especially in the early stages will provide important information regarding AD pathogenesis and help to design preventive and therapeutic reagents for the disease.